Association of Erythrocyte Hemolysis Products and Kidney Injury During Neonatal Cardiac Surgery

Abstract

Background/Objectives: Hemolysis has been associated with acute kidney injury (AKI) in infants and neonates after surgery involving cardiopulmonary bypass (CPB). Erythrocyte hemolysis and subsequent end-organ injury have been shown to be a complex process involving the liberation of multiple molecules that mediate the loss of nitric oxide and oxidative damage. This study assesses the association of multiple products of erythrocyte hemolysis with the evolution of AKI in neonates and infants undergoing CPB surgery. Methods: Blood and urine samples were collected at multiple time points before and after CPB and stored within an institutional biorepository. Twenty-one patients with AKI were matched with twenty-one non-Aki patients based on demographic and case complexity data. Results: Samples were analyzed for cell-free hemoglobin, heme, non-transferrin-bound iron, haptoglobin, hemopexin, and nitrite/nitrate. NGAL and KIM-1 were measured to index AKI. Cell-free hemoglobin was higher, haptoglobin was lower, and haptoglobin:hemoglobin ratio was lower in AKI compared to non-AKI patients. Conclusions: AKI in neonates and infants after CPB is associated with a pre and postoperative decrease in serum haptoglobin. These results confirm the need for future studies to prevent injury from hemolysis during CPB and potentially identify at-risk patients with decreased haptoglobin levels before surgery if delay is an option.

1. Introduction

Cardiopulmonary bypass (CPB) is required to provide adequate access for cardiovascular surgical repair in children born with congenital heart disease. The exposure of blood to mechanical stresses and non-biological surfaces of a CPB circuit has been clearly shown to cause hemolysis and inflammation [1,2]. CPB prime for smaller patients utilizes the addition of banked donor blood to achieve adequate hemoglobin (Hgb) levels during CPB, which enhances pathologic processes. Multi-organ dysfunction, including acute kidney injury (AKI) has been associated with this CPB inflammation.

AKI in children undergoing cardiac surgery is a common problem affecting up to 62% of patients in this high-risk group [3,4]. The etiology of AKI is multifactorial and likely includes low perfusion of the kidney during prolonged CPB [5], reperfusion injury, nephron-toxic drugs, and inflammation [6].

The risk of developing AKI may be enhanced by exposure to cell-free hemoglobin (CFH) during CPB [7] and this response is associated with a decrease in serum haptoglobin levels. The negative effects of chronic exposure to CFH are evidenced in microcirculatory dysfunction, thrombosis, vasculopathy, recurrent infections, mortality of sick cell disease, and transfusion of older stored red blood cells (RBC) [8]. Biochemical and molecular mechanisms underlying these effects include pro-oxidant reactions mediated by heme redox cycling and rapid scavenging of endogenous nitric oxide (NO). All of these properties may underlie CFH-mediated AKI [9].

In addition to CFH, free heme and low molecular weight iron-containing molecules are products of hemolysis that may act via distinct pathways to elicit AKI. CFH, heme, and non-transferrin-bound iron are all pro-oxidants, but likely act in different cellular and tissue compartments; free heme is hydrophobic for example. Moreover, free heme does not scavenge NO; however, it induces cell permeability, inhibits innate immunity, and promotes tissue inflammation via multiple pathways including toll-like receptor 4 pathways and inflammasome [10,11].

Finally, endogenous pathways that mitigate free Hgb and heme-dependent effects, namely the acute phase proteins haptoglobin and hemopexin, respectively, are critical modulators of potential toxicity mediated by hemolysis. An important consideration here is not only the levels of hemolysis products, haptoglobin, and hemopexin but relative amounts of haptoglobin to free Hgb and hemopexin to free heme. While associations between CFH and AKI have been reported, the roles of other hemolysis products, particularly in relation to endogenous heme and Hgb scavenging systems, in the development of AKI have not been characterized. We performed a comprehensive assessment of how products of hemolysis and metabolites of NO, namely nitrite and nitrate, change in neonates undergoing CPB and relate these AKI. The objective of our study was to confirm the association of CPB-induced hemolysis and AKI, and further identify if specific components produced from hemolysis were more closely associated with AKI or other postoperative outcome measures.

2. Materials and Methods

All chemicals, reagents, and assay kits were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) unless otherwise noted.

2.1. Study Design

The institutional review board at the University of Alabama at Birmingham approved this study, and informed consent was waived. Support for this research was provided by Phillippe Lathrop endowed chair fund in Pediatric Cardiac Anesthesiology. Plasma samples collected from neonates and infants (<30 days) undergoing cardiac surgery involving CPB October 2012–December 2018 and who consented to plasma and urine collection and storage in our institutional biorepository were surveyed. Plasma was collected via aspiration after centrifugation and stored at −80 °C. Urine was collected at the appropriate time point via Foley catheter and stored at −80 °C. AKI was defined as the Acute Kidney Injury Network (stage 2) as this was felt to represent significant injury. Other criteria for defining AKI are available and our selection is comparable to RIFLE (risk, injury, failure, loss of kidney function) criteria for injury or Kidney Disease Improving Global Outcomes (KDIGO) stage 2. Patients who developed AKI, and for whom samples collected immediately before CPB, immediately after CPB, 4 h, 24 h, and 48 h post-CPB were available, were randomly selected. Subject matching (21 AKI vs. 21 non-AKI) based on demographic and complexity metric (Matching criteria: The Society of Thoracic Surgeons-European Association for Cardio-Thoracic Surgery [STAT] category, CPB ± minutes, weight ± 0.5 kg).

2.2. Endpoints

The primary objective for this study was the correlation of serum levels of Hgb, haptoglobin, heme, etc., in patients with and without AKI as defined by increases in serum creatinine. Further data were retrospectively collected from patient electronic medical records and included weight, gender, age at the time of surgery, STAT category, CPB time, aortic cross-clamp (ACC) time, use of circulatory arrest, lowest preoperative creatinine, postoperative lactate, intraoperative hemodynamics, vasoactive-inotropic score (VIS) at 24 h post-admission, daily fluid balance, and postoperative creatinine levels.

The case type was also reviewed to ensure no statistical significance existed between groups comparing the number of specific cases such as arch reconstruction, Norwood procedure, Blalock-Taussig shunt, etc.

2.3. Pre-Operative and Intraoperative Management

Fluid management in the cardiovascular operating room included maintenance of intravenous fluid and blood products per the anesthesiologist’s discretion as indicated by clinical circumstances. The CPB circuit prime consisted of 25% albumin, fresh frozen plasma (20 mL/kg), packed RBCs, mannitol, sodium bicarbonate, and Normosol-R. Patients underwent zero-balance ultrafiltration during CPB and modified ultrafiltration at the end of CPB. Those requiring circulatory arrest were cooled to 22 °C and received one dose of DelNido cardioplegia. During arch reconstruction, continuous low-flow cerebral perfusion was employed. Post-CPB management was under the direction of the attending anesthesiologist, utilizing routine clinical protocols for hemodynamic and coagulation management targeting age and lesion-specific parameters. Standard hemodynamic management upon separation from CPB included low-dose epinephrine, milrinone, and vasopressin. Hemodynamic instability was initially managed with preload augmentation via colloid and blood product administration as opposed to frequent inotrope titration, such that hemodynamic instability in many patients is primarily reflected by increased volume administration in the immediate postoperative period. As described above, inotropic scores were analyzed between groups (AKI vs. non-AKI) to ensure no difference in clinical management existed.

2.4. Plasma Measurements

Plasma (300 µL) thawed on ice and the following measurements were made:

Non-Transferring Bound Iron (NTBI) Measurement

Plasma NTBI was measured with slight modifications as described previously [12,13]. Briefly, 250 µL of Iron (III) Chloride standard (0, 0.1, 0.5, 1, 5, 10, and 50 µM) or plasma were incubated with nitrilotriacetic acid (NTA, pH 7.0, final concentration 80 mM) for 30 min at 20–25 °C. Samples were then filtered using Centricon (3 kDa MW, Millipore, Billerica, MA, USA) at 10,620× g for 2 h at 15 °C. 200 µL of the eluant was acidified with hydrochloric acid (HCl) (final 5 mM) in 96 well plates and incubated for 30 min at room temperature. Iron-detection reagent (0.5 mM ferrozine, 0.5 mM neocuproine, 0.21 M ammonium acetate, and 0.07 M ascorbic acid in water, final concentrations) was then added for 1 h at room temperature. Ferrous-FerroZine complex was measured at 540 nm using a plate reader (BioTek, Winooski, VT, USA). Plasma NTBI concentrations were calculated by comparison to a standard curve generated using Iron (III) Chloride. The concentration of standards was verified using the extinction coefficient for Ferrous-Ferrazine complex at 562 nm (27.9 nM⁻¹ cm⁻¹).

2.5. Plasma Nitrite and Nitrate Measurement

Nitrite and Nitrate were extracted with a mixture of methanol (2:1, v:v) and centrifuged at 13,000 rpm for 10 min, 4 °C. Nitrite and nitrate concentrations were measured in methanolic extracts by high-performance liquid chromatography (HPLC)-coupled to the Griess reaction, and comparison to sodium nitrite and nitrate standards prepared daily (AMUZA Inc. San Diego, CA, USA) as described [14].

2.6. Plasma Haptoglobin and Hemopexin

Plasma haptoglobin and hemopexin concentrations were determined by enzyme-linked immunosorbent assay (ELISA) sandwich assay (Abcam, ab108858, and ab221838 respectively, Massachusetts, MA, USA) according to the manufacturer’s instructions.

2.7. Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Kidney Injury Molecule-1 (KIM-1)

Urine was available on 11/21 patients in AKI and non-AKI groups. Urine thawed on ice and NGAL and Kim-1 were determined by ELISA sandwich assay (Invitrogen, KIT036, Carlsbad, CA, USA, and R&D system, DKM100, Minneapolis, MN, USA) according to the manufacturer’s instructions.

2.8. Measurement of Hemoglobin and Heme

Plasma concentrations of cell-free oxyhemoglobin, cell-free methemoglobin, and CFH were measured by spectral deconvolution as described [15]. Absorbance spectra were measured at pH 7.4 and absorbance between 450 and 700 nm was measured using a Beckman UV-Vis Spectrophotometer at 1 nm intervals, using 1 mm path length cuvettes at room temperature. Standards for oxyhemoglobin, methemoglobin and cyanomethemoglobin, CFH, conjugated and unconjugated bilirubin were prepared in PB, pH 7.4. Data are reported as total Hgb (indicating all Hgb redox and ligation states), CFH, or total heme (i.e., Hgb + CFH). For CFH and total heme measurements, 17/104 samples were excluded due to sample turbidity which precludes measurement of CFH by this method. Two data points from hemopexin:CFH ratio were excluded based on the data outlier test.

2.9. Statistical Analysis

Descriptive analyses (means, standard deviations [SD or SEM], medians, interquartile ranges [IQR], and frequency distributions [%]) were performed to assess and describe study subjects. All data were assessed for normality using the Shapiro–Wilk test and appropriate post-tests were selected accordingly. Paired t-tests were performed for the assessment of changes within each group and Mann–Whitney U tests were performed to compare the area under the curves (AUC) between non-AKI and AKI patients. Wilcoxon signed-rank tests were used to compare non-normal continuous measures between groups. The two-way ANOVA mixed-effect model was assessed with Tukey’s multiple comparisons post-hoc test to assess differences between non-AKI and AKI patients’ time-dependent changes. Logistic regression models were used to identify independent risk factors (at time 24 h) associated with AKI and odds ratios (OR) with 95% confidence interval (CI) were estimated to quantify the effects of risk factors on AKI. To take into account correlated subjects or clustering of subjects due to matched paired data, robust estimates of standard errors were used. A complementary classification and regression tree (CART) analyses were also performed to identify predictors associated with AKI status. p-values < 0.05 were considered significant. All analyses were performed using GraphPad Prism Software (Version 10.1.2, San Diego, CA, USA), SAS version 9.4 (Cary, NC, USA), and R program version 4.0.3. Power and sample size: With a sample size of 40 (20 in each group) we have 83% power to detect an odds ratio of 0.35 associated with 1 unit increase in Haptoglobin at the significance level alpha = 0.05. Thus, we have adequate power to detect at least one biomarker associated with AKI. Power and sample estimates were performed by power and sample size software (PASS version 21).

3. Results

Table 1. Patient Demographics.

Demographics	AKI, Yes n = 21	AKI, No n = 21	p-Value
Age, days	7 (4, 9)	8 (6, 15)	0.15
CPB, minutes	107 (97, 121)	108 (88, 113)	0.84
ACC, minutes	62 (46, 70)	55 (41, 69)	0.79
Circulatory arrest, minutes	3.8 ± 10.3	5.1 ± 11.1	0.72
Low flow cerebral perfusion, minutes	8.9 ± 14.1	9.7 ± 16.6	0.86
Lowest preoperative creatinine, mg/dL	0.5 (0.4, 0.6)	0.5 (0.4, 0.6)	0.74
Weight, kg	3.2 (2.9, 3.5)	3.3 (2.9, 3.4)	0.97
Gender, Male (n (%))	16 (76.2)	14 (66.7)	0.73
STAT category, n (%)			1.00
3	6 (29)	6 (29)
4	13 (62)	13 (62)
5	2 (9)	2 (9)

Data presented as median (interquartile range (IQR)) or median ± standard deviation (SD). CPB, cardiopulmonary bypass; ACC, aortic cross-clamp; STAT, The Society of Thoracic. Surgeons-European Association for Cardio-Thoracic Surgery.

shows patient demographics. Figure 1A,B show levels of NGAL and KIM-1 in urine collected at 4 h or 24 h after CPB in patients who did or did not develop AKI defined by Acute Kidney Injury Network (AKIN) criteria. No changes in NGAL were observed; however, KIM-1 increased significantly in the AKI group confirming AKI diagnosis.

Figure 2A–E show that plasma CFh, plasma total heme, and plasma protein levels increased significantly immediately post-CPB in all patients and returned to basal within 24 h. No significant changes in plasma NTBI were observed, and with plasma CFH, levels were higher immediately post-CPB in Aki and non-AKI groups and remained elevated in the former, whereas they decreased 24–48 h post-CPB in non-AKI patients. Figure 2F–J show results from OAUC analysis and demonstrates significant differences between AKI and non-AKI groups were only observed for Hgb and total heme, with exposure being ~1.5 fold higher over the 48 h observations period.

Figure 3A shows in the non-AKI group, levels of plasma haptoglobin trended towards an increase immediately post-CPB, and then further increased with levels being significantly higher at 48 h. In the AKI group, following a similar increase immediately post-CPB, levels decreased at 24 h and then increased at 48 h. Haptoglobin prevents CFH toxicity via high affinity binding with one haptoglobin binding to one Hb-dimer. Figure 3B plots how the ratio of Haptoglobin-Hgb (dimer) changes post-CPB and AUC analysis. Immediately post-CPB, the Haptoglobin-Hgb ratio trended to decrease to a ratio of ~1 in both patient groups. This ratio rebounded and increased in non-AKI patients by 24 h and 48 h, whereas in AKI patients, the Haptoglobin-Hgb dimer ratio remained low at 24 h reaching basal levels by 48 h. Hemopexin levels increased immediately post-CPB and returned to baseline levels by 48 h, with no differences between AKI and non-AKI patients observed (Figure 3C). Similarly, Figure 3D shows that the hemopexin-CFH ratio increased in both non-AKI and AKI patients immediately post-CPB and quickly decreased by 4 h, reaching close to baseline levels by 24 h. Figure 3E–H, respectively, show AUC analyses for changes in haptoglobin and hemopexin shown in Figure 3D. Haptoglobin levels were lower in AKI patients (Figure 3E) with the Haptoglobin-Hgb dimer ratio being ~2.7-fold lower in AKI patients (Figure 3F).

Figure 4A shows that plasma nitrite levels were highest immediately post-CPB relative to 24 h for non-AKI patients. No changes were observed in AKI patients, and across 48 h no change in nitrite was observed between patients (Figure 4B). Plasma nitrate levels were lowest immediately post-CPB relative to 48 h for both AKI and non-AKI patients, with no significant changes across 48 h noted (Figure 4C,D).

Univariate logistic regression modeling for the outcome AKI (vs non-AKI) demonstrates that higher plasma total heme, lower haptoglobin levels, and lower haptoglobin:Hgb ratios were significantly associated with AKI (

Table 2. Biomarker predictors of acute kidney injury.

Predictors	Odds Ratio (95% CI)	AUC	p-Value
Total plasma heme (µM)	1.06 (1.001, 1.115)	0.78	0.0445
Plasma Haptoglobin, (mg/mL)	0.30 (0.10, 0.87)	0.72	0.0264
Ratio Hp:Hb dimer	0.66 (0.49, 0.88)	0.78	0.0054

CI, confidence interval; AUC, area under the curve; Hp, haptoglobin; Hb, hemoglobin.

). However, total Haptoglobin (µM):Hgb dimer (µM) was found to be independently associated (OR = 0.61, CI = 0.42–0.91, p = 0.0141) with AKI in multivariable logistic regression when adjusted for total plasma heme, excluding plasma haptoglobin from the model due to collinearity with total Haptoglobin (µM):Hgb dimer (µM). A complementary CART analysis with three predictors (Table 2) demonstrated the utility of Haptoglobin (µM):Hgb dimer (µM) and total plasma heme in predicting AKI.

Figure 5 demonstrates via CART analysis that subjects with Haptoglobin (µM):Hgb dimer (µM) ≥ 5.7 are non-AKI (11/12 = 92%) and those are <5.7 but total heme < 26 would predict also non-AKI (6/9 [67%]; specificity [11 + 6]/21 = 0.81) and those with Haptoglobin (µM):Hgb dimer (µM) < 5.7 but total heme ≥ 26 would predict AKI (17/21 [81%], sensitivity).

Further analyses included a comparison of clinical data and management parameters including hemodynamics, inotrope, blood product, or volume administration between groups. No significant differences were found.

4. Discussion

In this study, we confirm the association of higher levels of CFH and heme with Aki in pediatric-CPB and further show that the ratio of haptoglobin:Hgb may be an additional and more sensitive indicator of patients likely to develop AKI. Mechanical stresses associated with the transit of RBCs through CPB circuits lead to hemolysis and studies have linked released Hgb to adverse outcomes in these settings. Pediatric patients may be more susceptible to inflammatory and pro-oxidant effects of hemolysis owing to their limited ability to mount antioxidant and acute phase responses to pro-inflammatory/pro-oxidant stimuli. Indeed, previous studies have shown associations between Hgb and AKI in children undergoing CPB [7,16]. Our results confirm these earlier observations; patients who develop AKI were exposed to higher levels of Hgb over 48 h post-CPB. Although not significant, baseline (pre-CPB) levels of total heme were higher in patients who developed AKI. This may suggest that factors intrinsic to the patient may modulate hemolysis or propensity for RBCs to lyse independent of CPB per se. Additional studies with higher sample sizes are needed to further investigate this possibility. Furthermore, haptoglobin decreased within 24 h post-CPB in patients that subsequently developed AKI, but increased in non-AKI patients. This is consistent with exposure to higher Hgb concentrations and resultant consumption of haptoglobin; levels were higher at 48 h likely reflecting increased production of this acute phase protein. Importantly, when plotted as the ratio of haptoglobin:Hgb, it is clear that all patients show an initial drop in this ratio post-CPB, due to increased plasma Hgb levels. In patients that develop AKI, the ratio remains low over 24 h, whereas in patients that do not develop AKI, the ratio increases ~3-fold. Since the initial increase in Hgb post-CPB was similar in all patients, we speculate that a relatively lower haptoglobin response underlies the development of AKI. These data also underscore the importance of measuring not only the concentrations of Hgb and haptoglobin but also their ratio. It is possible that a high Hgb level may not lead to toxicity if a robust haptoglobin response ensues. Equally, it is possible that low Hgb levels lead to AKI if haptoglobin response is insufficient.

We also measured CFH and NTBI. Interestingly, only modest changes post-CPB were observed with these species, and no differences between non-AKI versus AKI patients. The primary product released from hemolysis is Hgb, with CFH and NTBI postulated to be released subsequently as Hgb undergoes denaturation and/or clearance. It is possible that changes in CFH and NTBI may be observed at later times; however, we have observed that in models of trauma and RBC resuscitation, both CFH and NTBI increase concomitantly with Hgb [10,17], suggesting that CPB itself, while damaging to RBC, does not lead to an environment that causes a significant breakdown of CFH. We also note that measured levels of CFH at baseline (~20 µM) were higher compared to healthy adults (<5 µM). Whether this reflects a normal situation in children or reflects underlying disease is not clear. Either way, this level of CFH has been shown to elicit cell permeability and injury [10,11,18]. Notably, hemopexin levels were higher in all patients immediately post-CPB to the extent that the ratio with CFH was above one, a condition expected to prevent CFH toxicity. Thus, we speculate that robust and early hemopexin response, coupled with a limited increase in CFH levels limit the role of this species in mediating end-organ injury in this patient setting. That said, the ratio of hemopexin to CFH decreased to below one at 24–48 h post-CPB. Whether this may contribute to other adverse outcomes including increased infection risk needs to be further tested. Similarly, future study groups defined by preoperative haptoglobin levels may further define associated risks.

Limitations of this study include a small sample size which is common due to limitations in case volume and patient enrollment in research studies. Further limitations include the length of time needed to acquire appropriate samples for our study design. Urine samples, pre- and post-CPB, were a significant limitation in obtaining an adequate number of patients to be tested for the markers investigated in this study. This limitation is multifactorial but includes low urine output, sample loss around collection catheters, and inadequate volume. These limitations, compounded with increased variance in analyte levels such as NGAL, could have led to a lack of difference found by other groups [16]. The study design was based on utilizing AKIN criteria for kidney injury, and certainly, other criteria for defining AKI are available. Selection of AKIN stage 2 is comparable to RIFLE “failure” criteria for injury or KIDGO stage 2.

5. Conclusions

Once outside the confines of the erythrocyte, CFH wreaks havoc on multiple tissues, leading to dysfunction, injury, and cell death. This understanding stems largely from hemolytic disease (e.g., sickle cell disease) and experiences with CFH-based blood substitutes [19,20,21]. More recent evidence suggests that hemolysis plays a role in non-hemolytic acuate inflammatory diseases, especially in critical care settings (e.g., sepsis, infection, stored RBC transfusion, and associated end-organ injury), leading to a renewed focus on understanding various species that are released during hemolysis and the mechanisms linking these to increased inflammation and oxidative tissue injury [18,22,23,24,25,26,27,28]. It is now clear that in addition to CFH, free heme and iron are all independent mediators of toxic effects associated with hemolysis.

In summary, our data supports previous findings that neonates and infants suffer a larger burden of AKI after cardiac surgery involving CPB. Further, this AKI is associated with increased exposure to products of erythrocyte hemolysis with the ability of a given subject to buffer this exposure with haptoglobin, likely playing an important role in whether AKI ensues or not. More studies are needed to understand the protection afforded by haptoglobin, and the utility of increased haptoglobin as therapeutic strategies to protect against AKI.